Many drug receptors are allosteric proteins and efficacy reflects the extent to which agents cause structural changes. The long term goal of this study is to understand the molecular nature of drug efficacy. The central hypothesis is that receptors posses a limited number of structural features by which allosteric agents influence their function. Just as drug interaction studies led to the identification of saturable binding sites, this study will look for evidence of "allosteric saturation" by studying the interactions between agents which act on the same receptor through distinct binding sites. The presence of a sufficiently efficacious agent should reduce the observed effect of a second agent if both affect a common structural feature. Studies will be conducted on the GABAA receptor which is a ligand-gated chloride channel with at least five separate allosteric modulator binding sites. Four structurally distinct modulators (phenobarbital, 5beta-pregnan-3alpha-o1-20-one,H+ and La3+) will be studied. Outside-out patches from HEK 293 cells transiently transfected with GABAA receptor genes will be exposed to rapid application of various concentrations of GABA in the presence or absence of a single modulator. The effect of each modulator on the kinetics of receptor activation, desensitization, deactivation and recovery from desensitization will be determined. Modulators will then be applied in combination to determine how the presence of one modulator affects the potency and efficacy of another. Results from this study can eventually be used to guide studies on mutated receptors in order to identify the structural features responsible for allosteric modulation. This study will also add to the understanding of the GABAA receptor which is the major inhibitory neurotransmitter receptor in the central nervous system and the site of action of many toxic and therapeutic agents.